Phosphatase assay using lyophilized aryl phosphate monesters

ABSTRACT

Lyophilized aryl phosphate monoesters having a pH of substantially 1 to 4 upon reconstitution with the amount of water removed during lyophilization; are prepared by lyophilizing an aqueous solution of the aryl phosphate monoesters having a pH of substantially 1 to 4. Diagnostic compositions for the determination of phosphates in which the lyophilized aryl phosphate monoesters are employed.

Unite States Patent [19] Hammer 1451 Feb. 12, 1974 1 PI-IOSPHATASE ASSAY USING LYOPHILIZEI) ARYL PHOSPHATE MONESTERS Related US. Application Data [62] Division of Ser. No. 856,205, Sept. 8, 1968, Pat. No.

Frank E. Hammer, Chicago, 111.

[52] US. Cl 195/1035 R I [51] Int. Cl. GOll'l 31/14 [58] Field of Search 195/1035 R [56] References Cited UNITED STATES PATENTS 3,425,912 2/1969 Deutsch et a1. 195/1035 R FOREIGN PATENTS OR APPLICATIONS 863,739 3/1961 Great Britain 1,153,109 5/1969 Great Britain 195/1035 R OTHER PUBLICATIONS Bessey et a1., Jour. Biol. Chem, 196:175-178 (1952).

Primary ExaminerA1vin E. Tannenholtz Assistant Examiner-Max D. Hensley Attorney, Agent, or FirmSybil Meloy [57] ABSTRACT Lyophilized aryl phosphate monoesters having a pH of substantially 1 to 4 upon reconstitution with the amount of water removed during Iyophilization; are prepared by lyophilizing an aqueous solution of the aryl phosphate monoesters having a pH of substantially 1 to 4. Diagnostic compositions for the determination of phosphates in which the lyophilized aryl phosphate monoesters are employed.

4 Claims, N0 Drawings PHOSPHATASE ASSAY USING LYOPHILIZED ARYL PHOSPHATE MONESTERS This is a division of application Ser. No. 856,205, filed Sept. 8, 1969, now US. Pat. 3,723,579.

The present invention relates to novel lyophilized aryl phosphate monesters, more particularly phenyl phosphate or p-nitrophenyl phosphate, which have a pH of substantially l to 4 upon reconstitution with that volume of water removed during lyophilization; the process for their preparation; and diagnostic compositions in which such a lyophilized monoester can be usefully employed for the determination of phosphatase.

Though phenyl phosphate and p-nitrophenyl phosphate can be employed for various chemical purposes, for example as chemical intermediates or as buffers, their widest use is in biochemistry as substrates in assay procedures designed to determine phosphatase, i.e.; phosphomonoesterase, levels in various body fluids. It is known, for example, that alternation of phosphatase levels occurs in certain diseased states such as, in particular carcinomas, and diseases associated with increased osteoblastic activity in bone and in obstructive, infiltrative, and parenchymal diseases of the liver and biliary tract. Additionally, the presence of phosphatase can be used to indicate the presence of certain kinds of body fluids, e.g. semen.

In general, the usefulness of aryl phosphate monoesters in the determination of phosphatase in clinical chemistry is based upon the fact that phosphatase will catalyze the hydrolysis of such aryl phosphates as phenyl phosphate and p-nitrophenyl phosphate. The hydrolysis reaction has been suggested to be depicted as follows:

Phosphatase is considered to catalyze the final step of the above hydrolysis. The term p-nitrophenyl phosphate and phenyl phosphate are defined in this applica tion to signify the free acid form, the monoionic form, and the diionic form represented by the foregoing general structures. The source of the p-nitrophenyl phosphate and phenyl phosphate is either the free acid,

ROH pO or an ammonium, alkali metal or alkaline earth metal salt thereof. Inthis application, the terms pnitrophenyl dihydrogen phosphate and phenyldihydrm gen phosphate specifically indicate the free acid which for the purposes of this invention is equivalent to the ammonium, alkali metal or alkaline earth metal salts thereof.

In clinical chemistry, phosphatases are referred to as acid or alkaline, the terms indicating the optimum pH at which they are measured. The usual procedure for the determination of either acid or alkaline phosphatase involves the incubation of a phosphate monester such as p-nitrophenyl phosphate or phenyl phosphate in an appropriately buffered medium with a sample of the fluid to be analyzed for the presence of phosphatase. The above depicted hydrolysis reaction is allowed to occur and thereafter the mixture then is analyzed either for inorganic phosphate or the presence of free or ganic alcohol, ROH, which comprised the organic portion of the phosphate monoester, to provide an indication of the levels of phosphatase present. Because the rate of hydrolysis is actually the determinative factor in the phosphatase assay, it is particularly important that the reactants be substantially unhydrolyzed prior to use in the assay. In the case of materials known to be prone to absorption of moisture, it is particularly important that such materials be applied in an anhydrous form in order to avoid the inhibition of the enzyme activity attributable to the partial hydrolysis accompanying such hydration. Though p-nitrophenyl dihydrogen phosphate and phenyl dihydrogen phosphate are reputed to be particularly convenient substrates for use in phosphatase determination, their proclivity for hydration and consequent hydrolysis limits their usefulness. Con sequently it was desired to prepare a phenyl dihydrogen phosphate and a p-nitrophenyl dihydrogen phosphate which would be resistant to hydrolysis and able to endure longer shelf life prior to use in a phosphatase assay.

It has been suprisingly found that preparing a solution of phenyl phosphate or p-nitrophenyl phosphate which has a pH of substantially l to 4 and most preferably a pH of substantially l, and subjecting that solution to lyophilization enhances the resistance to hydrolysis of the resultant lyophilized material. Such a preparation may be suitably achieved by mixing the components of phenyl dihydrogen phosphate or p-nitrophenyl dihydrogen phosphate or the alkali metal, ammonium or alkaline earth metal salts thereof in such porportions as to form a solution having a pH within the desired range of substantially l to 4 and preferably about pH 1. A solution of such pH range can also be prepared by dissolving a quantity of phenyl dihydrogen phosphate or p-nitrophenyl dihydrogen phosphate or an alkali metal, ammonium or alkaline earth metal salt thereof in water and adjusting the pH of the resultant solution. Most preferably, a solution of desired pH can be obtained by acidifying a quantity of water to a pH within the desired range and adding to that acidified solution a quantity of phenyl dihydrogen phosphate, p -nitrophenyl dihydrogen phosphate or an alkali metal, ammonium or alkaline earth metal salt thereof sufficient to dissolve in the acidic aqueous media without significantly changing the pH from the desired level. Any acid of sufficient strength may be used in the adjustment of the pH except that when the end product is destined for use in a phosphatase assay, acids which contribute anions which may interfere with enzymatic activity such as phosphoric acid or sulfuric acid should not be employed. The contemplated lyophiization can be desirably carried out in a freeze dryer (lyophilizer) at shelf temperatures of about 20 to 30C. for a period of 48 to 96 hours and most preferably at a temperature of about 25 for a period of about 48 to 72 hours. Suitable ranges of pressure are approximately to 25 microns while most preferably the pressure is maintained at about 25 microns. It will be apparent to one skilled in the art that lyophilization temperatures and pressures are interdependent, lower temperatures resulting in higher pressures. Of course, the lyophilization can be carried out at higher or lower temperatures with a commensurate loss in stability as a result of the tendency towards hydrolysis at higher temperatures and moistures, and extension of time of drying at lower temperatures. Most desirably, it has been found that the moisture of the matrix should be maintained at about 3 per cent since this provides good stability.

The pH of the solution to be lyophilized is critical. As previousy specified this pH must be substantially 1 to 4, with the most preferred pH being substantially l. The term substantially is used to indicate that the pH adjustment can be made to a point a few tenths ofa pH point below 0.5 or above 4 without unacceptably altering the amount of hydrolysis during and for extended periods after lyophilization. Thus when the hydrolysis was carried out at pH 5 an unacceptable change in absorbence at about 400 millimicrons of from about 0.09 to about 0.4, after storing the lyophilized material for 22 hours at 40 C., was observed. An unhydrolyzed material would show substantially no absorbence, ie less than 0.1, upon reconstitution with about 400 times the volume of water in the mixture prior to lyophilization. At a lyophilization pH of 8, absorbence immediately after lyophilization was an unacceptable 0.3 and after 22 hours at 40 C. was an even more unacceptable 0.94. At a lyophilization pH of 12, absorbence immediately after lyophilization was an unacceptable 0.74 and after 22 hours at 40 C. was more than 1.5.

The resistance to hydrolysis exhibited by the lyophilized product of the instant invention is particularly unexpected in view of the known fact that aqueous solutions of alkyl and aryl phosphate monoesters exhibit minimum or essentially no hydrolysis at a pH of around 7 or higher while greatest hydrolysis is observed at between pHs of almost to 6, with maximum velocity at about pH 4. However, we have found a completely opposite effect where frozen solids and lyophilization are concerned.

The lyophilized product obtained according to the procedure outlined above has the added advantage of being in the dry state which permits the material to be packaged in containers which are easy to handle, store e.g. at C. preferably in the absence oflight, and use. Further, each of the containers may include a quantity of the material which is exactly that amount required for making a particular number of phosphatase determinations or for a single phosphatase determination. In order to make one such phosphatase determination, the contents of one standard size container may be fixed with a predetermined quantity of aqueous buffer to produce a liquid reagent that is suitable for making a single assay. For the determination of phosphatase activity, it is necessary to use an acidic or alkaline buffer dependent upon whether acid or alkaline phosphatase is to be determined. The buffer selected should be characterized by a pH and pK of about the pH needed for maximum velocity of the enzyme reaction which is being assayed. An example ofa suitable buffer for alkaline phosphatase is 2-amino-2-methyl-lpropanol as illustrated in Example 6 following while a suitable buffer for acid phosphatase is illustrated by the citrate buffer exemplified in Example 7 following. Alternatively, the lyophilized product of this invention may be formed into tablets, pills or other forms which can provide diagnostic compositions which are usefully employed in the determination of phosphatase. Additionally, the diagnostic compositions of this invention can be adherent in an inert solid support material. For example, said compositions may be applied to splinters, sticks or strips made of wood fiber including paper, glass, metal or plastic. Particularly preferred are bibilous materials such as paper, wood fiber or the like having any desired shape which readily absorb a solution of the lyophilized material of this invention to form an impregnated bibilous mat of paper. Alternatively, it has been discovered that such bibilous strips may be impregnated with a solution of pnitrophenyl phosphate or phenyl phosphate which has been adjusted or prepared such that it exhibits a pH of substantially l to 4 without the need for lyophilization of this solution followed by reconstitution. Drying of the strip which has been impregnated with the substantially pH 1 to 4 solution provides a ready form of diagnostic composition which is characterized by enhanced stability, longer shelflife and convenient form for simple and accurate usage by the clinician.

As indicated above, the p-nitrophenyl dihydrogen phosphate and phenyl dihydrogen phosphate can be used in their free acid form as starting materials for the lyophilization process, but the ammonium, alkali metal and alkaline earth metal salts thereof are preferred from the standpoint of commercial availability. Moreover, the alkali metal salts such as lithium, potassium and especially sodium are particularly convenient for use. A most preferred embodiment of this invention is a lyophilized p-nitrophenyl phosphate which has been derived from disodium p-nitrophenyl phosphate by dissolution of that material in acidic aqueous media having a pH of substantially 1 followed by subjection to lyophilization. The product thus obtained upon reconstitution with the amount of water removed during lyophilization exhibits a pH of substantially l.

The free organic phosphate monoesters of this invention can be obtained by any standard procedure, such as the passage of an alkali metal salt through a cationic exchange resin, e.g. Dowex 50 in the acid form. The ammonium, alkali metal and alkaline earth metal salts can be obtained by the addition of an excess of the appropriate hydroxide to a solution of the free organic phosphate monoestcr, followed by evaporation of the solvent or by ion exchange using for example as a starting material the readily available sodium salt.

The following examples will further illustrate specific embodiments of the invention. These examples are set forth by way of illustration only and it will be understood that the invention is not to be construed as limited either in spirit or in scope by the details contained therein as many modifications and substitutions in both materials and methods will be apparent from this disclosure to those skilled in the art. Percentages refer to percent by weight unless otherwise indicated, relative amounts of materials are expressed in parts by weight, except as noted otherwise, and temperatures are given in degrees Centrigrade. The relationship between parts by weight and parts by volume is the same as that existing between grams and milliliters. The abbreviations used are as follows: mlliliters: ml. Molar: M; millimicrons: mp. for Wavelength of light; Normality: N.

EXAMPLE 1 Parts by volume of a pH 10.43 solution containing 9.075 parts of disodium p-nitrophenyl phosphate in deionized water was divided into 20 aliquots containing 5 parts each. To six of these aliquots was added sufficient hydrochloric acid to attain pH levels of 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 respectively, as determined by a pH meter. After pH adjustment, the solution was diluted to 6.75 parts by volume by the addition of deionized water. Six vials at each pH level were prepared containing 0.45 parts by volume of the above resulting solution and the vials were thereafter capped with rubber stoppers placed half way through the vial openings.

The liquid containing vials prepared above were placed in a freeze-dryer (lyophilizer) on shelves having a temperature of about -40 to about -50 C. After the liquid was frozen, vacuum was applied to the chamber until the pressure reached about 200 microns. At that point, the condenser was cooled and the shelves were heated to about 20 C (at this shelf temperature, the pressure in the chamber was about 50 microns and the temperature of the condenser was about 50 C). The vials were lyophilized for 72-96 hours under the above conditions. At the end of that time nitrogen was allowed to enter the chamber until 5 inches of vacuum was reached. The vials were then stoppered in the freeze-dryer with the stoppering plate provided therein. After removal from the freeze-dryer, the vials are further sealed by crimping aluminum seals over the stoppers. Upon reconstitution of the lyophilized material present in each of the vials prepared above, no appreciable change in pH was observed to have occurred during lyophilztion.

EXAMPLE 2 About 20 parts of water was adjusted to a pH of 1.1 i 0.05 with aqueous hydrochloric acid. To this solution was added 0.32 part of disodium p-nitrophenyl phosphatc. The resulting solution was determined to have a pH of 1.4. One part of volume of this solution was pipetted into a vial and subjected to the lyophilizatlon procedure described in the second paragraph of Example 1 except that shelf temperatures of about 20-30 C. and a lyophilization period of about 48-72 hours were employed rather than those described in Example 1. The resultant lyophilized material showed no appreciable absorbance at about 400 millimicrons when reconstituted with water.

EXAMPLE 3 100 Parts by volume of a solution containing 2 parts of dipotassium phenyl phosphate in deionized water was divided into 6 aliquots. To each of these aliquots was added sufficient hydrochloric acid to achieve pH levels of 0.5, 1.0, 1.5, 2.0, 3.0 and 4.0 respectively, as determined by a pH meter. After pH adjustment, the vials were treated identically with those prepared according to Example 1 and thereafter lyophilized according to the procedure described in the second paragraph of Example 1 to provide a stable lyophilized material.

EXAMPLE 4 An equivalent quantity of dimagnesium pnitrophenyl phosphate, was substituted for the disodium p-nitrophenyl phosphate in Example 2 and the procedures described therein are repeated. The lyophilized material thus obtained shows no appreciable change in absorbance at about 400 millimicrons when reconstituted with water.

EXAMPLE 5 An equivalent quantity of diammonium pnitrophenyl phosphate, was substituted for the disodium p-nitrophenyl phosphate in Example 2 and the procedures described therein are repeated.

EXAMPLE 6 Diagnostic Composition For The Determination of Alkaline Phosphatase.

1. Buffer: milliliters of 0.84 M 2-amino 2- methyl-l-propanol buffer having a pH of about 10.2.

2. Phosphate Substrate: Vials each containing 0.34 micromoles of the lyophilized disodium p-nitrophenylphosphate prepared according to Example 2 and 0.0105 micromoles of magnesium chloride are stored at refrigerated temperatures.

21 Ml. of the buffer was added to a vial containing the phosphate substrate, with mixing. This working reagent is stable for at least two months when refrigerated.

0.5 Ml. of this solution was added to each of two cuvettes, and the cuvettes placed in a water bath at 37 C. for 5 minutes. To one of the cuvettes was added with mixing 0.02 ml. of the specimen to be tested for phosphatase, e.g. serum or plasma; this cuvette serving as a test sample. The other cuvette to which no specimen was added at that time, served as a blank. The cuvettes were incubated at 37 C. for exactly 30 minutes. At the end of the incubation period, 5 ml. of 0.05 N sodium hydroxide, was added with mixing to each of the cuvettes. To the blank cuvette there was then added 0.02 ml. of the specimen.

Standards were prepared using a known concentra tion of p-nitrophenol, suitably 10 ml. of 0.36 micromoles.

The intensity of the color developed in the test samples and blanks was read at any of the wave lengths between 400 to 420 millimicrons in a colorimeter or spectrophotomer in which the absorbance of a water blank was set at zero. Standards are read at the same wave length used to read the test samples and the blanks. The actual absorbance of the test sample is calculated by subtracting the blank absorbance from the sample absorbance. The actual absorbance is compared with the reading obtained with the reference standard to estimate the amount of phosphatase in the specimen.

EXAMPLE 7 Preparation of diagnostic composition for the determination of acid phosphatase.

1. Buffer: A 0.22 M citrate buffer, of pH 4.8 is prepared by dissolving 42 grams of citric acid in 400 ml. of l N aqueous sodium hydroxide, then diluting the solution to 1,000 ml. with distilled water.

2. Phosphate substrate: 3 grams of the lyophilized disodium phenyl phosphate prepared according to Example 3 is dissolved in 250 milliliters of buffer prepared in the foregoing paragraph.

The test employing this diagnostic composition may be carried out in a manner conventional for acid phosphatase determination such as that described in the fol' lowing paragraph.

A mixture of l milliliter of the phenyl phosphate substrate, l milliliter of the citrate buffer and 0.2 milliliter of the specimen to be analyzed for acid phosphatase is incubated at 37 C. for exactly 5. minutes. At the end of the incubation period, the reaction is stopped and the mixture made alkaline by the addition of 0.375 N aqueous sodium hydroxide. To this mixture there is then added 1 milliliter of buffered aminoantipyrine solution (This solution can be prepared by mixing grams of 4-aminoantipyrine, 30 grams of sodium carbonate and 30 grams of sodium bicarbonate, then diluting with distilled water to a final volume of 1,000 milliliters.) with mixing, followed by the addition of l milliliter of buffered potassium ferricyanide solution (This solution is prepared by diluting a mixture of 40 grams of potassium ferricyanide, grams of sodium carbonate and grams of sodium bicarbonate to 1000 milliliters with distilled water) to result in the formation of a red solution the absorbance of which is measured at 505 millimicrons m;;.. A control sample identical to the incubation mixture except that no specimen is present is also subject to the above procedure, thus serving as the reagent.

The amount of phenol released during the incubation can be estimated with reference to working standards containing a known concentration of phenol and thus the activity of the acid phosphatase in the serum can be determined. Likewise, a control serum of known acid phosphatase activity can be used as a reference standard.

What is claimed is:

1. In a process for measuring phosphatase by contacting an aqueous buffered solution ofa phosphatase containing specimen with a phenylphosphate substrate and measuring a decomposition product released from the substrate by the action of the phosphatase, the improvement which comprises using phenylmonophosphate lyophilized at pH 1-4 as the substrate.

2. A process as in claim 1, wherein the substrate is lyophilized at pH 1.

3. A process as in claim 2, wherein the substrate is pnitrophenyl phosphate.

4. A process as in claim 1, wherein the lyophilized substrate is adhered to a solid support material. 

2. A process as in claim 1, wherein the substrate is lyophilized at pH
 1. 3. A process as in claim 2, wherein the substrate is p-nitrophenyl phosphate.
 4. A process as in claim 1, wherein the lyophilized substrate is adhered to a solid support material. 